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. 2020 Jul 21;9(7):1740.
doi: 10.3390/cells9071740.

The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain

Tabea Osthues  1 , Béla Zimmer  2 , Vittoria Rimola  2 , Kevin Klann  3 , Karin Schilling  2 , Praveen Mathoor  4 , Carlo Angioni  2 , Andreas Weigert  4 , Gerd Geisslinger  1   2 , Christian Münch  3   5 , Klaus Scholich  1   2 , Marco Sisignano  1   2
Affiliations

The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain

Tabea Osthues et al. Cells. .

Abstract

Nerve injury-induced neuropathic pain is difficult to treat and mechanistically characterized by strong neuroimmune interactions, involving signaling lipids that act via specific G-protein coupled receptors. Here, we investigated the role of the signaling lipid receptor G2A (GPR132) in nerve injury-induced neuropathic pain using the robust spared nerve injury (SNI) mouse model. We found that the concentrations of the G2A agonist 9-HODE (9-Hydroxyoctadecadienoic acid) are strongly increased at the site of nerve injury during neuropathic pain. Moreover, G2A-deficient mice show a strong reduction of mechanical hypersensitivity after nerve injury. This phenotype is accompanied by a massive reduction of invading macrophages and neutrophils in G2A-deficient mice and a strongly reduced release of the proalgesic mediators TNFα, IL-6 and VEGF at the site of injury. Using a global proteome analysis to identify the underlying signaling pathways, we found that G2A activation in macrophages initiates MyD88-PI3K-AKT signaling and transient MMP9 release to trigger cytoskeleton remodeling and migration. We conclude that G2A-deficiency reduces inflammatory responses by decreasing the number of immune cells and the release of proinflammatory cytokines and growth factors at the site of nerve injury. Inhibiting the G2A receptor after nerve injury may reduce immune cell-mediated peripheral sensitization and may thus ameliorate neuropathic pain.

Keywords: 9-HODE; G2A; GPR132; macrophage migration; neuropathic pain; oxidized linoleic acid metabolites.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
GPR132 (G2A)-deficiency leads to reduced mechanical hypersensitivity and the secretion of oxidized lipids is affected in wild-type mice. (A) Paw withdraw latency (PWL) of ipsilateral site of wild-type mice (WT, triangle), G2A-deficient mice (G2A−/−, squares) and sham-treated mice (circles) after spared-nerve injury (SNI) surgery. n = 10 animals, male and female, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 (WT vs. G2A−/−); ### p < 0.005, #### p < 0.0001 (WT vs. sham); $$ p < 0.01, $$$ p < 0.005, $$$$p < 0.0001 (G2A−/− vs. sham). Statistics were performed with two-way Analysis of variance (ANOVA) with Bonferroni correction. (B) Schematic depiction of the oxidative linoleic acid pathway. (C,D) Concentrations of 9- hydroxyoctadecadienoic acid (HODE) in sciatic nerve (SN), L4–L6-dorsal root ganglia (DRG) and spinal cord (SC) in wild-type (C) and G2A-deficient (G2A−/−) (D) mice 7 days after SNI surgery. (E, F) Concentrations of 13-HODE in sciatic nerve (SN), dorsal root ganglia (DRG) and spinal cord (SC) in wild-type (E) and G2A-deficient (G2A−/−) (F) mice 7 days after SNI surgery. (G–J) Concentrations of epoxyoctadecenoic acids (EpOMEs) and dihydroxyocatadecenoic acids (DiHOMEs) in SN (G,H) and DRG (I,J) in wild-type (G,I) and G2A−/− (H,J) mice 7 days after SNI surgery. Black represents untreated site (contralateral). Grey represents treated site (ipsilateral) in the respective tissues from n = 5 mice per group, male and female. Data represent mean ± standard error ofmean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.005; Two-way ANOVA with the Holm-Sidak method.
Figure 2
Figure 2
Reduced number of immune cells in G2A-deficient mice 7 days after SNI surgery. (A) Total immune cell number at the ipsi- and contra-lateral site of sciatic nerve (SN) (CD45+). (B,C) Number of different types of immune cells in the ipsi- and contra-lateral site of the sciatic nerve. (D,E) Immunohistochemical staining of macrophages (F4/80, CD11b) at the injured sciatic nerve (SN) in wild-type (D) and G2A-deficient mice (E) 7 days after SNI. Dashed lines indicate sites of injury. (F) Total immune cell number in L4-L6-DRGs at the ipsi- and contra-lateral site of sciatic nerve. (G,H) Number of different types of immune cells in the ipsi- and contra-lateral site of L4–L6-DRGs 7 days after SNI surgery. (I) Total immune cell number in the dorsal spinal cord receiving input from spinal cord comparing the ipsi- and contra-lateral site. (J,K) Number of different types of immune cells in the dorsal spinal cord section receiving input from L4–L6-DRGs comparing the ipsi- and contra-lateral site 7 days after SNI surgery. Data were obtained from n = 5 animals per group, male and female. Ipsilateral site of WT mice is shown in black. Ipsilateral site in SN is shown in yellow, in DRG in purple and in SC in green. Contralateral site of WT mice is depicted in dark grey. Contralateral site of G2A-/- mice is shown in light grey. Neutrophils (CD45+, Ly6G+, CD11b+), macrophages (CD45+, Ly6G−, CD11b+, F4-80+, Ly6C−), monocytes (CD45+, Ly6G−, CD11b+, F4-80−, Ly6C+), dendritic cells (DC; CD45+, Ly6G−, CD11b+, F4-80−, Ly6C−, CD11c+, MHCII+), CD11b+ NK cells (CD45+, Ly6G−, CD11b+, F4-80−, Ly6C−, NK1.1+), NK cells (CD45+, Ly6G−, CD11b−, F4-80−, Ly6C−, NK1.1+), T cells (CD45+, Ly6G−, CD11b−, F4-80−, CD3+, MHCII−), CD4 T cells (CD45+, Ly6G−, CD11b−, F4-80−, CD3+, MHCII−, CD4+), CD8 T cells (CD45+, Ly6G−, CD11b-, F4-80−, CD3+, MHCII−, CD8+), B cells (CD45+, Ly6G−, CD11b−, F4-80−, CD11c−, CD3−, MHCII+, CD19+). Data represents mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; Two-way ANOVA with Bonferroni correction.
Figure 3
Figure 3
Reduced concentrations of inflammatory cytokines in G2A-deficient mice. (A) Heat map of cytokines levels in sciatic nerve, DRG and spinal cord at the ipsilateral site of wild-type and G2A-deficient mice 7 days after SNI. Data shown as ipsi-lateral of WT vs. ipsi-lateral of G2A-defcient mice measured with LUMINEX. (B–F) Concentrations of tumor necrosis factor α (TNFα) (B), interleukin 6 (IL-6) (C), nerve growth factor (NGF) (D), transforming growth factor β(TGFβ) (E) and IL-1β (F) in the ipsilateral site of sciatic nerve 7 days after SNI in wild-type (WT, black) and G2A-deficient mice (G2A−/−, yellow in sciatic nerve), measured with enzyme-linked immunosorbent assay (ELISA). Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 of n = 5 mice per group, male and female; one-way ANOVA.
Figure 4
Figure 4
Protein regulation in wild-type bone marrow-derived macrophages (BMDMs) 24 h after stimulation with 9-HODE. (A) Relative mRNA expression of G2A receptor in BMDMs treated with 1 µM 9-HODE for 24 h. n = 6–10 animals per group, male. (B) Identification of >6.000 regulated proteins 24 h after stimulation with 1 µM 9-HODE in BMDMs. Fold change (FC) [log2] is plotted against p-values [Log10]. Significant downregulated proteins are depicted in blue, and red represents upregulated proteins. (C) Percentage of downregulated proteins 24 h after 1 µM 9-HODE stimulation compared to untreated BMDMs. (D) Percentage of upregulated proteins 24 h after 1 µM 9-HODE stimulation compared to untreated BMDMs, clustered in groups. yellow: immune system, blue: lipid metabolic process, brown: adhesion, green: migration, grey: apoptosis, pink: lysosome, red: inflammation, white: hematopoiesis, black: other. Data represents mean ± SEM. * p < 0.05. An unpaired one-tailed t-test was used for statistics.
Figure 5
Figure 5
Transient increase of matrix-metalloproteinase 9 (MMP9) secretion and MMP9 downregulation in BMDMs 24 h after G2A activation. (A) List of the strongest upregulated migratory proteins in WT 24 h after 1 µM 9-HODE stimulation. (B) Comparison of the strongest upregulated migratory proteins in WT and G2A-deficient (G2A−/−) BMDMs regarding the toll-like receptor 4 (TLR4)-signaling pathway shown as -log p-value. (C) Scheme of possible G2A–TLR4 interaction and signaling in BMDMs. (D) List of the 10 strongest downregulated migratory proteins in WT 24 h after 1 µM 9-HODE stimulation. (E) Representative Western Blots of MMP9 expression and respective analyzed data in untreated and treated BMDMs with 1 µM 9-HODE. (F) MMP9 expression in BMDM lysates treated with 1 µM 9-HODE for different time points. (G) MMP9 secretion of BMDMs treated with 1 µM 9-HODE for different time points. Analyzed with ELISA. n = 6–10 male animals per group. Data represents mean ± SEM. * p < 0.05, ** p < 0.01. An unpaired one-tailed t-test and a One-way ANOVA were used.
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